Throughout this application, various patents and papers are referenced. The disclosures of these patents and papers in their entireties are hereby incorporated by reference into this application in order to more fully describe the state-of-the-art as known to those skilled therein as of the date of the invention described and claimed herein.
This invention relates to a method for discriminating white blood cells from red blood cells and platelets in unlysed, whole blood by use of a red-fluorescent dye and flow cytometry techniques.
The need to supply the clinical environment with automated instrumentation has forced the development of a wide variety of instruments capable of performing various types of blood cell counts. Automated counting of red blood cells (erythrocytes), platelets and leucocytes (white blood cells) may be accomplished by a variety of techniques. One class of instruments for use in counting blood cells includes those based on flow cytometry principles.
Flow cytometry combines many of the advantages of microscopy and biochemical analysis in a single high precision technique for the rapid analysis and sorting of individual cells. The cells to be measured by this technique are introduced into the center of a fast moving fluid stream and forced to flow single file out of a small diameter orifice at uniform speeds. The particles are hydrodynamically focused to the center of the stream by a surrounding layer of sheath fluid. The cells within the stream pass a measurement station where they are illuminated by a light source and measurements are made at rates of 2.5.times.10.sup.2 to 10.sup.6 cells per minute. Laser light sources are used in the measurement of the cells. Typical laser light sources used include argon ion lasers (UV, blue and green light), krypton lasers (yellow and red light), helium-cadmium lasers (UV and blue light), and helium-neon lasers (red light).
When a cell in the flow stream passes through the light beam, the illuminating light is scattered by the cell and the intensity of scatter at different angles yields information about cell size and surface morphology. Although the light is scattered in all directions, the intensity of light scattered at low angles in the forward direction along the axis of the illuminating laser beam (forward angle light scatter; FALS) is related to the cell size. For example, lymphocytes have little FALS and can be typically distinguished from larger granulocytes with a greater FALS, while monocytes display FALS values which are about the same as granulocytes.
The light scattered by the cells and collected orthogonally to the laser beam (90.degree. right angle or wide-angle light scatter) mostly represents light reflected from the internal or surface structures of the cell and is interpreted as an index of cellular granularity. Use of the combination of FALS and right-angle scatter parameters is more reliable than either parameter alone in discriminating different classes of human blood cells. In addition to these two parameters, fluorochromes may also be used to label the cells of interest. The fluorescence emitted by the cells when excited by the illuminating laser beam yields additional information about the cells for distinguishing subpopulations of cells.
U.S. Pat. No. 3,883,247 (Adams) describes a composition and method for the differential analysis of white blood cells into six categories, namely lymphocytes, monocytes, neutrophils, eosinophils, basophils and immature granulocytes. The white blood cells are treated under conditions in which the cells are "shocked" by exposure to a non-physiological medium, namely a hypotonic aqueous salt solution, during staining with the metachromatic fluorochrome dye acridine orange. The method involves suspending a fresh blood sample in the hypotonic aqueous acridine orange solution for a time period and then subjecting the suspension to radiation from a blue laser. The cells are then differentially classified on the basis of the differences in the magnitudes of red and green fluorescence emitted from individual cells in response to excitation from the blue laser radiation.
A problem with the method taught by Adams is that it requires the use of an argon-ion laser for providing radiation having a wavelength in the blue spectrum for exciting the acridine orange fluorophor. The argon-ion laser is an expensive light source and it would be much more desirable to utilize a light source that provides radiation in the red spectrum, such as the helium-neon laser. An additional problem of the Adams method, is that it only uses the parameter of fluorescence to differentiate the white blood cells. As such, there is no means for distinguishing cells based on cell size, such as the FALS parameter used in flow cytometry techniques. Thus, the Adams method is not suitable for quantitating and differentiating red blood cells, platelets and the sub-populations of white blood cells all in one procedure.
Further problems exist with the method taught by Adams. The use of the hypotonic diluent causes the size of the red blood cells to change. This is a disadvantage when an accurate size measurement of the cells is needed to provide an accurate quantitation of the cells. Also, the red cells absorb the acridine orange thereby changing the effective concentration of the dye in the sample. This results in less dye available to stain the white cells. This problem is difficult to alleviate because of the narrow range of effectiveness of the acridine orange. Use of too high a concentration of the dye results in all the cells absorbing a maximum amount of dye thereby preventing differentiation of the cells. Use of too low a concentration of the dye results in no staining at all.
It is an object of the present invention to provide a method for discriminating white blood cells from red blood cells and platelets as well as quantitating the red blood cells, platelets and white blood cells. A white blood cells differential of at least four-parts is obtained in a single procedure using flow cytometry techniques. It is an additional object of the present invention to achieve the differential of the white blood cells by use of a red fluorescent dye, such as an oxazine dye, having a wide effective range of concentration which is capable of being excited by an inexpensive light source such as a helium-neon laser.
The assignee hereof presently commercially offers the ELT series of instruments which are capable of providing red blood cell, white blood cell and platelet counts. Additionally, the following five traditional parameters are also provided: HGB (hemoglobin), HCT (hematocrit), MCV (mean cell volume), MCH (mean cell hemoglobin) and MCHC (mean cell hemoglobin concentration). The ELT series provides a three-part differential white blood cell count, namely lymphocytes, granulocytes and monocytes. There has been demand for an instrument which can provide a four-part or a five-part differential of white blood cells by further differentiating the granulocytes into neutrophils, basophils and eosinophils.
The prior methods which achieved a three-part differential of white blood cells required a two-step dilution of the patient blood sample in which the red blood cells were lysed. Lysis was required because the prior methods could not distinguish white blood cells from red blood cells. The red blood cells were lysed in order to remove them from the sample and prevent interference with white blood cell analysis. A major shortcoming of the lysis method is that some red blood cells are lysis resistant. Additionally, the lysing reagent also either lyses white blood cells or deleteriously affects the scatter characteristics of the white blood cells. Such results disadvantageously affect the accuracy of the cell counts.
U.S. Pat. No. 4,284,412 (Hansen et al.) describes an automated method for enumerating subclasses of blood cells in a blood sample. The method involves lysis of the red blood cells in the sample to be analyzed and then counting leucocyte subclasses, namely lymphocytes, monocytes and granulocytes. A flow cytometry system is used to measure right angle scatter, forward angle scatter and green fluorescence values. An FITC label and an argon-ion laser are used to generate the green fluorescence values.
U.S Pat. Nos. 3,916,205 and 4,146,604 (both in the name of Kleinerman) describe a method and composition for the counting of leucocytes, erythrocytes, reticulocytes and the differential counting and classification of leucocytes. A dye composition containing three different types of dyes (ethidium bromide, brilliant sulfaflavine and a stilbene disulfonic acid derivative) is described for distinguishing lymphocytes, monocytes, neutrophils and eosinophils. The method involves preparing an alcohol fixed blood smear on a glass slide. The blood smear is irradiated with three different light sources (UV, violet and green) in order to detect the differential staining of the cells.
Shapiro et al. used the same dye composition developed by Kleinerman in a flow cytophotometer to obtain a five-part differential of white blood cells, namely lymphocytes, monocytes, neutrophils, eosinophils and basophils (Shapiro, H. M. et al., J. Hist. and Cyt. (1976), 24: 396-411; Shapiro, H. M., J. Hist. and Cyt. (1977), 25: 976-989). The method described involves first fixing a blood sample with glutaraldehyde in an isotonic solution while avoiding lysis of the cells in order to enhance dye uptake. The dye composition is added after the cells are fixed and is then further diluted to enable an optimal cell sampling rate to be obtained in the cytophotometer. The parameters of forward angle scatter, right angle scatter and three different levels of fluorescence are measured. Three different lasers are used to irradiate the sample.
U.S Pat. No. 4,376,820 (Giannini et al.) describes a method for the quantitative evaluation of the total number of leucocytes and the number of granulocytes, monocytes and lymphocytes in a blood sample. The method involves forming an adduction of the leucocytes, an oxazine dye and a quencher molecule in a isotonic aqueous solution. The resultant adduct is subjected to radiation by a first ray of pulse light and then with a second ray of monochromatic light. The exit optical intensity of the latter ray of light is analyzed as a function of time. Before treatment of the cells, the blood sample is separated into white cells and red cells or the white cells are first concentrated.
U.S. Pat. No. 4,400,370 (Kass) describes a manual technique for analyzing human blood wherein a 5-part differential of white blood cells is achieved. The method involves contacting a whole blood sample with the dye basic orange #21 to distinguish lymphocytes, neutrophils, eosinophils, basophils and monocytes. This patent describes the use of one unique dye, namely basic orange #21, and no other dyes are exemplified. The patent describes that the dye used must be metachromatic in order to achieve the differentiation of the cells. It is suggested that the class of oxazines have a few species which would be operative in the method described. The method described requires manual microscopic analysis of cell morphology and therefore, does not work in a flow cytometry system.
U.S. Pat. No. 4,463,099 (Baroncelli et al.) describes the use of oxazines as immunofluorescent reagents. The immunofluorescent reagent described is prepared by reacting a protein to be labelled, a cross-linking agent and an oxazine dye.